Project Summary/Abstract Delayed-onset drug hypersensitivity reactions (DHR) are a significantly under-recognized and under- researched public health problem. Approximately 7% of the general population is affected by drug allergy. Skin is the most commonly affected organ and severity ranges from a mild rash to severe blistering and sloughing with internal organ involvement. The pathobiology of delayed-onset DHR is poorly understood due to a lack of both usable clinical samples and mouse models that recapitulate human disease. This negatively impacts patient care for 3 reasons. First, clinicians are ill-equipped to treat severe forms of disease. Second, there are no effective assays to identify a culprit drug, which can result in administering a less effective, more dangerous, and/or more-expensive drug in the future. Third, it can be difficult to discern drug allergy from other diagnoses with negative bearing on patient prognosis and/or treatment. It is therefore of paramount importance to elucidate the pathobiology of delayed-onset DHR to address these clinical needs. The central hypothesis of this project is that a recently identified population of T cells, termed skin resident-memory T cells (TRM), mediate delayed-onset DHR. The proposed project will directly test this hypothesis while overcoming current limitations in the field. Aim 1 employs three novel technologies to study a large bank of formalin-fixed paraffin embedded clinical specimens that until now were of little research utility. These technologies are (i) PerkinElmer?s OpalTM multiplexed immunohistochemistry and MantraTM Quantitative Pathology Workstation with inFORM Image Analysis Software, (ii) NanoString technology for gene expression profiling and (iii) ImmunoSEQ high-throughput TCR deep sequencing to assess TCR repertoire. Aim 1 further includes laboratory investigation of prospectively collected clinical specimens, by taking advantage of the patient volume of Harvard?s tertiary care hospitals. Aim 2 tests the role of skin TRM in delayed- onset DHR ex vivo, and in-so-doing develops a much needed assay to diagnose drug allergy. Aim 3 leverages progress in humanized mouse technology to study human skin TRM in an in vivo model of delayed-onset DHR, thereby creating a mouse model of disease for future research. The data generated through this project will advance both the fields of skin T cell biology and delayed-onset DHR.